1. Field of the Invention
The present invention is directed to the production of immortalized human corneal epithelial cell lines and their use.
2. Description of the Related Art
The corneal epithelium provides the characteristic epithelial barrier for the eye. It is a transparent barrier which also aides in maintaining the transparency of the underlying corneal stroma. The transparency allows light penetration to the retina and is crucial to visual acuity. Injury of the corneal epithelium may result in the loss of sight. Accordingly, to protect against inadvertent damage from commercially applied materials, the Food, Drug and Cosmetic Act of 1938 has required testing of cosmetic and drug products.
The current method for testing ocular irritancy in humans employs the use of rabbits. The method involves placing a foreign substance directly into the conjunctival sac of the rabbit eye. This assay is known as the Draize Test. The Draize Test is simple to perform, allows a quick economical result and uses a laboratory animal which is easy to breed and maintain. The test is described in Draize et al, 1944, J. Pharmacol. Exp. Ther. Vol. 82, pp. 377-390. However, there are drawbacks to the use of the Draize Test. First, there are morphological and biological differences between the laboratory animal eye and the human eye. See, for example, Goldberg, A. M. Ed., Alternative Methods in Toxicology: A Critical Evaluation of Alternatives to Acute Ocular Irritation Testing, Mary Ann Liebert, Inc., 1987. Secondly, in vitro models must satisfy several stringent criteria in order to be informative, including species and tissue specificity, which are key in order to determine specific biochemical and tissue specific mechanisms. Furthermore, animal rights groups adamantly protest against the use of animals in experimental research.
In vitro models for human corneal epithelium that utilize continuous cell lines have been proposed for toxicology studies. These models include the SIRC cell line (rabbit origin) which has a fibroblast morphology (Neiderkorn et al, 1990, In Vitro Cell Dev. Biol., Vol. 26, pp. 923-930) and the MDCK (Madin-Darby canine kidney) line (American Type Culture Collection. Catalog of Strains. 6th Ed., 1988, p. 21).
Alternatives to animal models have been proposed and human corneal organ culture techniques have been developed. See, for example, Doughman, D. J., 1980, Trans. Am. Ophthalmol. Soc., LXXCVIII, pp. 527-628; Richard et al, Current Eye Research, 1991, Vol. 10, pp. 739-749. In fact, primary cultures of human corneal epithelium have been used to model the ocular surface in vitro. See, Newsome et al, Invest. Ophthal., 1974, Vol. 13, p. 23; Ebato et al, Invest. Ophthal. and Vis. Sci., 1987, Vol. 28, pp. 1450-1456; and Hainsworth et al, Tissue Culture Methods, 1991, Vol. 13, pp. 45-48. However, primary cultures, even when maintained with fetal bovine serum, fibroblast feeder layers or growth supplements, become senescent after several passages in vitro. Thus, such cultures have a restricted finite life-span. In addition, not only is there biological variability among individual donors, but the availability of donor corneal material is uncertain.
There is therefore a need in the art for a substantially stable, continuous human corneal epithelial cell line. Such a cell line would be useful in the study of the effects of chemicals and drugs on the human eye as well as in basic research on the human eye. The present invention addresses this need by providing such a cell line derived from human corneal epithelial cells.